鸭疫里默氏杆菌RPA-LFD检测方法的建立与应用  

Establishment and application of RPA-LFD detection method for Riemerella anatipestifer

作　　者：葛佳仪 顾庆林 王碧 龙宥茨 李彩玲 吴琴 GE Jia-yi;GU Qing-lin;WANG Bi;LONG Yong-ci;LI Cai-ling;WU Qin(College of Animal Science,Guizhou University,Guiyang 550025,China;Institute of Animal Disease Research of Guizhou,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病研究所,贵州贵阳550025

出　　处：《中国预防兽医学报》2024年第11期1146-1152,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：贵州省大学生创新创业训练资助项目(gzusc2023032);贵州省农业攻关资助项目(黔科合支撑[2016]2506号);贵州大学基础研究项目(贵大基础[2023]12号)。

摘　　要：为建立一种便捷、新型、可视化,并能够用于现地检测鸭疫里默氏杆菌(RA)的方法,本研究经PCR扩增RA ompA基因并克隆至pMD19-T载体中,构建重组质粒pMD19-T-ompA,经PCR和测序鉴定正确后作为重组质粒标准品。按照TwistAmp^(R)nfo试剂盒说明书并采用TwistAmp^(R)nfo原理和横向流动试纸条(LFD)要求,根据RA ompA基因的保守区设计一对引物和一条探针。以pMD19-T-ompA质粒为模板,采用TwistAmp^(R)扩增试剂盒推荐的反应体系和条件经重组酶聚合酶扩增后,将LFD插入扩增产物中,于5 min之内观察结果,并利用单一变量法优化反应条件。结果显示,该方法在37℃反应15 min即可完成扩增,并且可以通过LFD可视化。表明初步建立了检测RA的重组酶聚合酶扩增横向流动试纸条(RPA-LFD)方法。以RA、E.coli、鸭源沙门菌(SE)、鸭源多杀性巴氏杆菌(Pm)、葡萄球菌(Staphylococcus)、鹅细小病毒(GPV)、鸭瘟病毒(DPV)、番鸭细小病毒(MDPV)的DNA作为模板,利用本实验建立的RPA-LFD方法检测,结果显示,除RA和质粒标准品pMD19-T-ompA在检测线出现红色条带为阳性结果外,其他病原均仅在质控线出现蓝色条带,未在检测线出现条带,均为阴性结果,表明该方法的特异性较强;以1.83×10^(7)拷贝/μL~1.83×10^(0)拷贝/μL的质粒标准品为模板,利用该RPA-LFD方法检测,结果显示该方法的检测限为1.83×10^(1)拷贝/μL,是环介导等温扩增横向流动试纸条(LAMP-LFD)方法检测限的1/10,比常规PCR方法高100倍,敏感性较高。利用建立的RPA-LFD方法和常规PCR方法检测64份临床鸭病料样品,结果显示2种方法对RA的检出率均为10.93%(7/64),二者的阳性符合率与总符合率均达100%。本实验建立的RPA-LFD方法特异性强、敏感性高、快速、准确,不需要昂贵仪器及专业人员,并能通过LFD使结果可视化,可适用于基层对RA的检测,为RA临床样品的检测和流行病学调查提供了一种新的技术手段。To establish a convenient,novel,visualizable method for on-site detection of Rimeria anatipestipestia(RA),the ompA gene fragment was amplified and cloned into the pMD19-T vector,and the resulting plasmid pMD19-T-ompA was confirmed by PCR and sequencing and was used as a standard plasmid.Following the guidelines of TwistAmp^(R)nfo amplification kit and in accordance with the TwistAmp^(R)nfo principle as well as the specifications for lateral flow strips(LFD),a pair of primers and a probe were designed based on the conserved region of the RA ompA gene.Using pMD19-T-ompA plasmid as a template,the recombinase polymerase amplification(RPA)was performed under the recommended conditions of the TwistAmp^(R)amplification kit.The results could be observed within 5 minutes after LFD was placed in the RPA product.The RPA-LFD method for detecting RA was initially established by optimizing the reaction conditions.This method is capable of accomplishing amplification at 37℃within 15 minutes and allows for visual interpretation via LFD.Subsequently,the established RPA-LFD method was applied for detection using the genomic DNA of RA,E.coli,Salmonella enterica(SE),Pasteurella multocida(Pm),Staphylococcus aureus,goose parvovirus(GPV),duck distemper virus(DPV),and Muscovy duck parvovirus(MDPV)as templates.The results showed that,except for RA and the plasmid standard pMD19-T-ompA,which showed a red band at the detection line as a positive result,all pathogens showed only a blue band at the control line,without a band at the detection line,indicating that the method had a strong specificity.Using the plasmid standard with a copy number ranging from 1.83×10^(7)copies/μL to 1.83×10^(1)copies/μL as a template,the results showed that the detection limit of the RPA-LFD method was 1.83×10^(1)copies/μL,which was 1/10 lower than that of the LAMP-LFD method and 100times higher than that of conventional PCR,indicating a high sensitivity.The established RPA-LFD method and conventional PCR method were used to detect 64 clinical duck tiss

关 键 词：鸭疫里默氏杆菌 ompA基因 重组酶聚合酶扩增 横向流动试纸条 

分 类 号：S852.61[农业科学—基础兽医学]